Despite advances in our knowledge of the causes and risk factors associated with periodontitis, there are no signs of a decline in its prevalence. Indeed, longer retention of teeth, coupled with an aging population might lead to future increases in the number of subjects affected by periodontal destruction. The severe limitations of the present clinical methods of diagnosis, result in an inability to distinguish subjects and sites at risk for periodontal disease initiation and progression. Thus, patients are often inappropriately treated. Analyses of biochemical markers of periodontal disease in GCF samples may overcome such limitations and provide clinicians with diagnostic and prognostic biomarkers of disease. The goal of the present investigation is to provide clinicians with a discriminatory and reliable analysis to help to diagnose disease status in individual subjects and sites. Specific Aim 1 will optimize and validate the checkerboard immunoblotting (CBIB) technique to quantify multiple components of the GCF, in a large number of samples. The parallel examination of several biomarkers in a large number of samples could result in a test with more significant diagnostic value. Moreover, the ability to investigate several molecules at once would allow inferences on the mechanisms of modulation of the immune response and tissue destruction by these mediators. Once a sensitive assay has been optimized, a cross-sectional pilot study will be performed as a first step in selecting possible biomarkers of disease progression. In Specific Aim 2, GCF samples will be obtained from 30 periodontally healthy and 30 periodontitis subjects in order to assess the diagnostic properties of the different GCF biomarkers in distinguishing health and disease on a subject and site level. Once the best candidate markers have been determined, the use of combinations of markers for improving diagnostic ability will be evaluated. The ultimate goal of this line of research is to conduct longitudinal studies on levels of GCF biomarkers, in order to determine their value as predictors of disease activity, and to better understand their cross-regulation. This goal is in accord with NIDCR's mission to identify markers of disease and elucidate biological events associated with inflammation, with the hope that this knowledge will lead to new therapies in the treatment of periodontal diseases. Moreover, this methodology is at the forefront of new approaches in proteomics, and carries enormous promise for translational applications in the near future. [unreadable] [unreadable]